Project Summary/Abstract: Cardiac optogenetics is an emerging technology with significant promise for the treatment of heart disease. Optogenetic actuators are ion channels or pumps that can be regulated by light, thus permitting the control of cellular electrical activity with high spatial and temporal precision. Optogenetics can be used not only to elucidate the electrical and mechanical functions of cardiac cells and heart tissue, but to potentially restore or repair malfunctioning pacemaker and conduction tissue. Unlike electrical implantable pacemakers, optogenetic actuators may permit optical modulation of electrical activity of cardiac pacemaker and conduction cells. Thus, this technology holds untapped potential for therapeutic applications. Significant hurdles must be overcome before this technology can be applied to human patients. The majority of optogenetic actuators operate in the 450-600 nm range, and light at these wavelengths is strongly scattered by cardiac tissue and absorbed by hemoglobin. These effects severely limit the tissue depth at which currently available optogenetic actuators can be used, thereby restricting their in vivo use to small rodents. To overcome this barrier, the use of longer- wavelength opsins has recently been suggested for optogenetics. The newest red-shifted opsins can operate at considerably greater tissue depths than conventional blue-sensitive channel-rhodopsins. Yet, even the most red-shifted opsins have significant overlap with the absorption spectrum of hemoglobin. Thus, an approach that could extend the operative range of actuators into the near-infrared (> 700 nm; i.e., beyond the hemoglobin absorption window) would be a major advance. The goal of this project is to co-express optogenetic actuators with a newly discovered enzyme, Cyp27c1, that can extend the spectral range of existing red-shifted optogenetic actuators into the near-infrared, thereby permitting optogenetic applications in the human heart. Cyp27c1 is used by fish and amphibians to extend their vision into the near-infrared in murky freshwater environments. The enzyme mediates this red-shift by adding an additional conjugated double bond to the visual chromophore, 11-cis retinal. The Corbo lab has demonstrated that Cyp27c1 can similarly act upon all-trans retinal, the chromophore of optogenetic actuators, to produce 3,4-didehydroretinal. Furthermore, a recent study showed that in vitro substitution of retinal with 3,4-didehydroretinal in optogenetic actuators can red-shift the actuator's action spectrum, underscoring the value of developing an in vivo strategy for cardiac applications.